The long-term goal of this project is to understand the cellular and molecular signals instigated in the testis that lead to germ cell apoptosis as a result of environmental toxicant-induced Sertoli cell injury. Since analogous decreases in Sertoli cell support and germ cell apoptosis occur during early postnatal testis development, revealing the mechanisms by which germ cell apoptosis is regulated will enhance our understanding of both the physiological importance of this process during distinctive periods of testicular development as well as to provide insights into cellular targets of environmental toxicants and possible mechanisms of infertility. We have previously revealed the participation of FasL and Fas, two members of the tumor necrosis factor (TNF) superfamily of proteins, in triggering apoptosis of specific germ cell sub- types after exposure to the Sertoli cell toxicant mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of the widely dispersed environmental agent DEHP. Here we propose to use this established Sertoli cell injury model to decipher the mechanism(s) responsible for conferring the unique sensitivity of spermatocytes/early round spermatids to undergo apoptosis after MEHP exposure. Our intriguing preliminary findings show that a soluble form of TNF-? (sTNF-?) is elicited by germ cells, via the action of distinct metalloproteinase (MP) enzymes, after MEHP-treatment and that sTNF-? may act upon TNFR1 on Sertoli cells to stimulate an increase in their expression of FasL. Our preliminary findings further indicate that Sertoli cells regulate the activity of MPs via their secretion of TIMP (tissue inhibitor of metalloproteinase) proteins into the adluminal space. Finally, the sensitivity of germ cells to FasL-mediated apoptosis is largely dependent on the ubiquitination, and subsequent degradation, of the anti-apoptotic protein c-FLIP. Our preliminary data suggest that the E3 ligase, Itch, ubiquitinates c-FLIP in germ cells secondary to MEHP- induced Sertoli cell injury. Taken together, these findings led to the development of the central hypothesis of this research proposal that decreases in Sertoli cell-derived TIMP proteins into the adluminal space, and the consequent activation of MPs, is a critical key event that instigates ensuing changes in specific germ cell sub-types that accounts for their sensitivity to undergo apoptosis during periods of reduced Sertoli cell supportive capacity. The first specific aim is designed to delineate the MP and TIMP family members in the testes and assess their role in the regulation of germ cell apoptosis. In the second aim, the functional participation of sTNF-? in FasL-mediated germ cell apoptosis will be challenged. In the last aim, experiments are focused on revealing the mechanisms regulating the levels of the c-FLIP protein and its role in altering the sensitivity of germ cell sub-types to undergo FasL-triggered apoptosis. PUBLIC HEALTH RELEVANCE: The focus of this grant project is to decipher the molecular and cellular mechanisms that regulate the death of testicular germ cells by the process of apoptosis. Apoptosis is an active process of cell death characterized by sequential phases of chromatin condensation, fragmentation and cell disintegration that leads to the orderly destruction and disposal of a cell without a consequent inflammatory response. Although many studies in humans have associated alterations in the incidence of germ cell apoptosis with conditions of abnormal spermatogenesis and male infertility, the cellular processes that regulate germ cell apoptosis in the testis remain poorly characterized. It is anticipated that the mechanistic insights provided from this work will be useful for predicting and preventing human reproductive health risks to chemicals, such as the phthalates, found ubiquitously in the environment.